There is growing evidence that activation of specific small GTPases regulates both increases and decreases in endothelial barrier permeability. Two novel GTPase dependent mechanisms to strengthen adhesion between endothelial cells, and thereby maintain normal permeability and attenuate both acute and chronic increase in microvessel permeability are the primary focus of this renewal application. These are: (1) the activation of Rapt by cAMP stimulated, but PKA independent, mechanisms;and (2) the activation of Rac1 by the novel anti-inflammatory agent, sphingosine-1-phosphate (S1P). At least one new therapeutic agent acting via Rac1 is currently in clinical trials as an immunosuppressant, and this project is designed to advance our understanding of the conditions where similar agents may be useful for anti-inflammatory therapies. The overall hypothesis is that regulation of permeability under different inflammatory conditions is determined by the balance of GTPase dependent mechanisms that strengthen adhesion between adjacent endothelial cells, stabilizing the selective barrier between blood and tissue, versus GTPase mechanisms that decrease adhesion and increase tension, forming inflammatory gaps between the endothelial cells. Aim 1 is to determine whether activation of the GTPase Rap1 by cAMP to strengthen cell-cell adhesion accounts for all the action of cAMP to maintain normal microvessel permeability before and after an acute increase in permeability. Aim 2 is to determine whether activation of the GTPase Rac1 by S1P to attenuate acute permeability increases is modified by competing mechanisms to increase permeability that are themselves Rac1 or S1P dependent. Aim 3 is to determine whether activation of Rap1 and Rac1 is less effective to attenuate increased permeability in microvessels exposed to inflammatory conditions for at least 6 hours than in normal microvessels. Venular microvessels in the rat mesentery and in skeletal muscle in control and transgenic mice will be cannulated and perfused to measure permeability properties before and after exposure to inflammatory conditions for at least 6 hours. Attenuation of increased permeability by Rap1 and Rac1 dependent mechanisms will be measured as well as changes in the distribution of key intercellular junction proteins by immunolabeling and the ultrastructure of intercellular junctions by serial section reconstructions. These methods are the most direct experimental approach to new understanding of regulation of microvessel permeability by small GTPases and to further evaluation of these novel targets for anti-inflammatory therapies directed at endothelial cell function.